Combination of fenofibrate and coenzyme q10 for the treatment of endothelial dysfunction

ABSTRACT

The present invention relates to a combination of a peroxisome proliferator activated receptor (PPAR) activator and a benzoquinone and their use in treating and/or preventing disorders characterized by endothelial dysfunction, such as cardiovascular disease, strokes and myocardial infarction. According to a preferred embodiment of the invention the benzoquinone or precursor thereof is a ubiquinone or precursor thereof, more preferably, coenzyme Q 10  or a precursor thereof, and the PPAR activator is a fibrate or a thiazolidinedione, more preferably fenofibrate.

This application is a 371 of PCT/EP01/12425 filed Oct. 23, 2001.

FIELD OF THE INVENTION

The present invention relates to a combination of a peroxisomeproliferator activated receptor (PPAR) activator and a benzoquinone andtheir use in treating and/or preventing disorders characterized byendothelial dysfunction, such as cardiovascular disease, strokes andmyocardial infarction.

BACKGROUND TO THE INVENTION

The burden of cardiovascular disease is increasing in both developed anddeveloping countries. This relates to an acceleration in the incidenceof diabetes and obesity as well as to other cardiovascular risk factors,including hypercholesterolaemia, hypertension and smoking. All theseconditions have in common a mechanism of vascular abnormality termedendothelial dysfunction (Rubanyi, 1993).

Nitric oxide (NO), a chemically unstable radical formed by enzymaticconversion of L-arginine in the presence of molecular oxygen, elicitsrelaxation of vascular smooth muscle cells. NO also counteracts plateletadhesion and aggregation. NO is released from endothelial cells by theaction of acetylcholine (ACh). Failure of the vascular endothelium toelicit NO-mediated vasodilatation may be due to decreased formation ofNO, increased degradation of NO and/or decreased biological sensitivityto NO. Irrespective of the mechanism this is referred to as endothelialdysfunction.

The vascular endothelium is also the site of formation of othervasodilator agents (e.g. prostacyclin, endothelium-derivedhyperpolarizing factor), as well as vasoconstrictive factors (e.g.thromboxane A2, endothelin).

Endothelium dysfunction is highly relevant to vascular disease andoccurs chiefly as a consequence of disturbances in the L-arginine/NOpathway.

Its occurrence in type 2 diabetes, for example, is extensively supportedby both in vitro and in vivo studies (Cohen, 1993; Watts, 1998). Indeed,endothelial dysfunction may be the initiating event in the process ofatherosclerosis eventually resulting in clinical coronary arterydisease. In hypercholesterolemic subjects, impairedendothelium-dependent vasodilatation is evidenced before the developmentof atherosclerosis. In patients with type 2 diabetes endothelialfunction is abnormal even in the absence of elevated plasma LDLcholesterol concentration.

Endothelial dysfunction in diabetes may have implications not only forcoronary artery disease, but also for peripheral vascular disease andretinopathy. Experimental and clinical studies support the concept thatdyslipidemia (in particular increased circulatory concentrations ofmodified, small dense LDL), as well as hyperoxidative stress, areclosely related to the development of endothelial dysfunction as aconsequence of changes in the disposal of nitric oxide NO.

Oxidative stress represents a challenge to normal bodily functions. Itmay arise from an increase in exposure to free radicals/oxidants or maybe a result of a decrease in anti-oxidant capacity. Oxidative stress iscaused by reactive oxygen species which can be of both endogenous orexogenous origin. Endogenous sources of free radicals, such as thesuperoxide anion O₂ ^(.−), include endothelial cells, activatedneutrophils and mitochondria. The term reactive oxygen species includesnot only oxygen-centred radicals (e.g. superoxide and hydroxyl), butalso non-radical derivatives of oxygen (H₂O₂), singulet oxygen and HOCl.In diabetes, as well as in myocardial infarction, stroke andinflammation, there is an increase in plasma levels of lipidhydroperoxides which are formed through a free radical-mediatedmechanism from polyunsaturated fatty acids.

Accordingly, given the association between oxidative stress, endothelialdysfunction and a range of important disorders there is a need toprovide an effective treatment for endothelial dysfunction caused byoxidative stress. In particular, type 2 diabetes is associated with amarkedly increased risk of cardiovascular disease, its majorcomplication.

Treatments have not been shown to be effective. There is a major needfor new preventative and therapeutic strategies for cardiovasculardisease.

DISCLOSURE OF THE INVENTION

Accordingly, the present invention provides a composition comprising aperoxisome proliferator activated receptor (PPAR) activator and abenzoquinone of formula I:

in which:

R1, R2 and R3 independently are:

-   -   an alkyl group having 1 to 8 carbon atoms, or    -   an alcoxy group having 1 to 8 carbon atoms;

R4 is:

-   -   an hydrogen atom,    -   an hydrocarbyl group having 1 to 60 carbon atoms,    -   a OR5 radical,    -   an SR6 radical,    -   a N(R7)(R8) radical,    -   a nitro group, or    -   a carboxyl group;

R5, R6, R7 and R8 being independently:

-   -   a hydrogen atom, or    -   an alkyl group having 1 to 20 carbon atoms;        or a precursor thereof capable of being metabolized in the human        or animal body to said benzoquinone;        or a pharmaceutically acceptable salt thereof.

In one preferred embodiment of the invention, said benzoquinone is ofthe formula (I) in which R₄ is an alkenyl group or a polyalkenyl group,preferably a group of formula:

in which n is an integer of from 1 to 12, preferably from 6 to 11.

Preferably the benzoquinone or precursor thereof is a ubiquinone orprecursor thereof, more preferably, coenzyme Q₁₀ or a precursor thereof.

Preferably, the PPAR activator is a PPARα or a PPARγ activator.

Preferably, the PPAR activator is a fibrate or a thiazolidinedione, morepreferably fenofibrate.

The PPAR activator, such as fenofibrate, may be co-micronised with asolid surfactant. Preferably, the solid surfactant is sodium laurylsulphate.

The present invention also provides a pharmaceutical compositioncomprising a composition of the invention together with apharmaceutically acceptable carrier or diluent.

The present invention further provides a method of treating orpreventing a disorder characterized by endothelial dysfunction in anindividual which method comprises administering to said individual aneffective amount of a peroxisome proliferator activated receptor (PPAR)activator and a benzoquinone of formula I or a precursor thereof capableof being metabolized in the human or animal body.

Typically, the disorder is selected from cardiovascular disease,hypertension, stroke, myocardial infarction, peripheral vasculardisease, angina pectoris, cardiac failure, diastolic and/or systolicventricular dysfunction, macro and microangiopathy in patients withdiabetes, and tissue damage related to ischemia or reperfusion.

The PPAR activator and benzoquinone may, for example, be administeredseparately, sequentially or concomitantly.

The present invention also provides a peroxisome proliferator activatedreceptor (PPAR) activator and a benzoquinone of formula I, or aprecursor thereof capable of being metabolized in the human or animalbody to said benzoquinone, for use in therapy.

Further, the present invention provides the use of a peroxisomeproliferator activated receptor (PPAR) activator and a benzoquinone offormula I, or a precursor thereof capable of being metabolized in thehuman or animal body to said benzoquinone, in the manufacture of amedicament for use in treating a disorder characterized by endothelialdysfunction, as defined above.

Further still, the present invention provides a method for producing acomposition of the invention which method comprises admixing said PPARactivator and benzoquinone.

The present invention also provides a method for producing apharmaceutical composition of the invention which method comprisesadmixing said PPAR activator and benzoquinone with a pharmaceuticallyacceptable carrier or diluent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the acetylcholine percent change in forearmblood flow ratio as a result of the administration of coenzyme Q₁₀and/or fenofibrate; and

FIG. 2 is a graph showing the sodium nitroprusside percent change inforearm blood flow ratio as a result of the administration of coenzymeQ₁₀ and/or fenofibrate.

BEST MODE(S) FOR CARRYING OUT THE INVENTION

Throughout the specification, unless the context requires otherwise, theword “comprise” or variations such as “comprises” or “comprising”, willbe understood to imply the inclusion of a stated integer or group ofintegers but not the exclusion of any other integer or group ofintegers.

PPAR Activators

The peroxisome proliferator activated receptor (PPAR) (Issemann, 1990)is a member of the family of ligand-activated nuclear receptorsincluding the estrogen receptor, the retinoic acid receptor (RXR) andthe androgen receptor. These nuclear receptors are activated by thebinding of a ligand, for example, estrogen, in the case of the estrogenreceptor. The activation of the receptor enables the latter to then bindto a specific DNA sequence, termed the responsive element, in thepromoter of a given gene leading thus to either an increase or in somecases a decrease in the transcription of the target gene.

PPAR is present as 2 main subtypes, PPARα and PPARγ. Both subtypes donot bind alone to the DNA promoter but must first dimerize with RXR.This heterodimer, composed of either PPARα and RXR or PPARγ and RXR thenbinds to a specific DNA sequence in the promoter, the peroxisomeproliferator responsive element. The endogenous ligands for PPARα andPPARγ are not known but are thought to be long chain fatty acids and/ortheir metabolites (Keller, 1993). PPARα and PPARγ control the expressionof genes involved in fatty acid and energy utilisation.

PPAR activators according to the present invention are activators ofPPARα and PPARγ. A number of PPAR activators are known in the artincluding the fibrate and thiazolidinedione classes of drugs, for whichfenofibrate and rosiglitazone, respectively, are well known examples.Activators of PPARα and PPARγ have overlapping as well as distinctpharmacological effects. In humans as well as in animal models,activation of PPARα with a fibrate, such as fenofibrate, or PPARγ withrosiglitazone leads to comparable lowering of serum triglycerides. BothPPARα and PPARγ are expressed in muscle, while PPARα is preferentiallyexpressed in hepatocytes and PPARγ in adipocytes. Fibrates mainlyactivate PPARα but bezafibrate has been shown to activate both PPARα andPPARγ. Similarly, rosiglitazone, an activator of PPARγ can also modifythe expression of genes normally controlled by PPARα.

Preferred PPAR activators according to the present invention areagonists of PPARα activity. It is particularly preferred to usefibrates, such as fenofibrate. A further example of a member of thefibrate family is given in U.S. Pat. No. 6,028,109.

Benzoquinones

Benzoquinones for use in the present invention are a benzoquinone offormula I:

in which:

R1, R2 and R3 independently are

-   -   an alkyl group having 1 to 8 carbon atoms, or    -   an alcoxy group having 1 to 8 carbon atoms;

R4 is:

-   -   an hydrogen atom,    -   an hydrocarbyl group having 1 to 60 carbon atoms,    -   a OR5 radical,    -   an SR6 radical,    -   a N(R7)(R8) radical,    -   a nitro group, or    -   a carboxyl group; or

R5, R6, R7 and R8 being independently:

-   -   a hydrogen atom,    -   an alkyl group having 1 to 20 carbon atoms;        or a precursor thereof capable of being metabolized in the human        or animal body to said benzoquinone;        or a pharmaceutically acceptable salt thereof.

In the description and the claims, the term “hydrocarbyl” is understoodas meaning an organic group comprising at least C and H. If thehydrocarbyl group comprises more than one C, then those carbon atoms maybe linked to each other directly by a single, double or triple bond, orindirectly by the intermediary of a suitable element or hetero atomssuch as for example oxygen, sulfur or nitrogen atoms or a suitable groupsuch as a carbonyl group.

The hydrocarbyl group may optionally comprise one or more suitablesubstituants. Examples of such substituants may include a halogen atom,an alkyl group having 1 to 8 carbon atoms, an alcoxy group having 1 to 8carbon atoms, a nitro group, a bis alkyl group having 1 to 16 carbonatoms, a cyclic group having 1 to 16 carbon atoms, etc.

The hydrocarbyl group may be any one of an alkyl group, an alcoxy group,a polyalcoxy group, an aryl group, an acyl group, an alkenyl group, apolyalkenyl group, including combinations thereof (e.g. an arylalkylgroup), which group may optionally contain one or more hetero atoms orone or more substituants on the chain or rings, as defined above.

The hydrocarbyl group is preferably a hydrocarbon group. Here the term“hydrocarbon” means any one of an alkyl group, an alkenyl group, analkynyl group, which groups may be linear, branched or cyclic, or anaryl group, or combinations thereof (e.g. an arylalkyl group). The termhydrocarbon also includes those groups but wherein they have beenoptionally substituted. If the hydrocarbon is a branched structurehaving substituent(s) thereon, then the substitution may be on eitherthe hydrocarbon backbone or on the branch; alternatively thesubstitutions may be on the hydrocarbon backbone and on the branch.

In one preferred embodiment of the invention R1, R2 and R3 are eachindependently a lower alkyl group or a lower alkoxy group. The term“lower alkyl group” means a straight-chain or branched alkyl grouphaving 1 to 8 carbon atoms, and examples thereof include methyl, ethyl,propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl(amyl), isopentyl, neopentyl, tert-pentyl, 1-methylbutyl, 2-methylbutyl,3-methylbutyl, 1,2-dimethylpropyl, hexyl, isohexyl, 1-methylpentyl,2-methylpentyl, 3-methylpentyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl,2,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl,3,3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, 1,1,2-trimethylpropyl,1,2,2-trimethylpropyl, 1-ethyl-1-methylpropyl, 1-ethyl-2-methylpropyland octyl groups. Among them, methyl, ethyl, propyl, isopropyl groups,etc., are preferable.

The term “lower alkoxy group” means a lower alkoxy group derived fromthe above-described lower alkyl group, such as methoxy, ethoxy andn-propoxy groups. Among them, a methoxy group is most preferred.

Preferably, R4 is an alkenyl group or a polyalkenyl group, i.e. a grouphaving one or more double bonds in any portion of an alkyl group. R4 maycomprise from 1 to 12, such as 6 to 11, preferably 9 or 10 repeats of anisoprenoid unit, such as 3-methyl-2-butene-1,4 diyl unit.

Preferably, R5, R6, R7 and R8 may each independently be H or C₁ to C4alkyl.

Compounds of the present invention may contain one or more asymmetriccarbon atoms and/or one or more non-aromatic carbon-carbon double bondsand may therefore exist in two or more stereoisomeric forms. Thus, thepresent invention also provides individual stereoisomers of thecompounds of the formula (I), as well as mixtures thereof, includingcompositions comprising the same.

Separation or diastereoisomers or cis and trans isomers may be achievedby conventional techniques, e.g. by fractional crystallisation,chromatography or HPLC of a stereoisomeric mixture of a compound of theformula (I) or a suitable salt or derivative thereof. An individualenantiomer of a compound of the formula (I) may also be prepared from acorresponding optically pure intermediate or by resolution, such as byHPLC of a racemate using a suitable chiral support or by fractionalcrystallisation of the diastereoisomeric salts formed by reaction of aracemate with a suitable optically active acid or base.

Benzoquinones for use in the therapeutic methods of the presentinvention should have antioxidant properties, such as the ability toscavenge active oxygen species. In addition, benzoquinones for use inthe therapeutic methods of the present invention will clearly need to bephysiologically acceptable upon administration and not cause excessiveside effects. For example, they should not be unduly toxic to patients.The toxicity of benzoquinones may be determined using a variety ofmethods known in the art including in vitro whole cell assays and LD₅₀animal tests.

U.S. Pat. No. 5,229,385, for example, describes a range of benzoquinonederivatives having antioxidant properties which may be usedtherapeutically. EP-A-419905 also describes a number of benzoquinonederivatives suitable for therapeutic use.

It will be appreciated by the skilled person that since the benzoquinonecoenzyme Q₁₀ is synthesised in vivo from precursor molecules, it may bepossible to administer a benzoquinone according to the present inventionby means of a precursor that is capable of being converted to abenzoquinone by the same biosynthetic pathways that produce coenzymeQ₁₀. Typically, a precursor will be an immediate precursor, that is tosay a molecule structurally related to the benzoquinone and that needsto undergo only a small number of steps in the biosynthetic pathwaybefore it is converted to a benzoquinone of formula I. It is generallypreferred to administer precursors that are processed by parts of thecoenzyme Q₁₀ biosynthetic pathway which are unique to coenzyme Q. Forexample, chorismate is converted in the body to p-aminobutyric acid,p-hydroxybenzoic acid, prephenate (which leads to phenylalanine andtyrosine) and is therefore not an immediate precursor since it suppliesseveral pathways. By contrast, p-hydroxybenzoic acid is used only in thesynthesis of ubiquinones and may be considered to be an immediateprecursor.

The skilled person will appreciate that the reason for preferringimmediate precursors is to avoid effects on other biosythetic pathwayswhich may have a deleterious effect on the patient.

The skilled person will also appreciate that the benzoquinones accordingto the present invention are reduced in the human or animal body to abenzoquinol. Consequently, since it may be possible to administerbenzoquinones in their reduced or oxidised state, references tobenzoquinones throughout mean both the quinone and reduced quinol forms.

It is particularly preferred to use coenzyme Q, a naturally occurringagent which acts as an electron carrier in the mitochondrial electrontransfer in the respiratory chain and which possesses several otherfunctions. CoQ is synthesised from condensation of a benzoquinone ringand a hydrophobic side chain varying in size between species withelongation through a trans-prenyl transferase with multiple repeats ofisopentenyl diphosphate units. In humans the side chain is composed often such repeats, that is the origin of its designation as CoQ₁₀.

In vivo the oxidized CoQ₁₀ is converted to reduced CoQ₁₀H₂ orubiquinol-10, a potent antioxidant in plasma, in lipoproteins and intissues. It scavenges in plasma free radicals produced by lipidperoxidation. CoQ₁₀ treatment has been previously demonstrated to besafe at doses up to 300 mg daily for the patient and in many countriesdifferent presentations are available over the counter. From previousevidence and as confirmed herein, CoQ₁₀ plasma levels increase 3 to 4fold after administration of 200 mg daily.

Administration

The amount of PPAR activator and benzoquinone or precursor thereof whichis required to achieve the desired biological effect will, of course,depend on a number of factors, for example, the mode of administrationand the precise clinical condition of the recipient. The followingroutes of administration and dosages described are intended only as aguide since a skilled practitioner will be able to determine readily theoptimum route of administration and dosage for any particular patientand condition.

In general, the daily dose of each component will be in the range of 0.1mg-100 mg/kg, typically 0.1-20 mg/kg. An intravenous dose may, forexample, be in the range of 0.01 mg to 0.1 g/kg, typically 0.01 mg to 10mg/kg, which may conveniently be administered as an infusion of from 0.1μg to 1 mg, per minute. Infusion fluids suitable for this purpose maycontain, for example, from 0.01 μg to 0.1 mg, per milliliter. Unit dosesmay contain, for example, from 0.1 μg to 1 g of each component. Thusampoules for injection may contain, for example, from 0.1 μg to 0.1 gand orally administrable unit dose formulations, such as tablets orcapsules, may contain, for example, from 0.1 mg to 1 g.

Preferably, the PPAR activator, particularly fenofibrate, isadministered in an amount from about 50 to 450 mg daily and thebenzoquinone or precursor thereof is administered in an amount fromabout 10 to 400 mg daily.

The PPAR activator and benzoquinone or precursor thereof may beadministered as the compounds per se, but are preferably presented withan acceptable carrier or diluent in the form of a pharmaceuticalcomposition. The carrier or diluent may be a solid or a liquid, or both,and is preferably formulated with the activator and benzoquinone as aunit-dose formulation, for example, a tablet, which may contain from0.05% to 95% by weight of the active component.

The formulations include those suitable for oral, rectal, topical,buccal (e.g. sublingual) and parenteral (e.g. subcutaneous,intramuscular, intradermal or intravenous) administration.

Formulations suitable for oral administration may be presented indiscrete units, such as capsules, cachets, lozenges or tablets, eachcontaining a predetermined amount of a PPAR activator and/orbenzoquinone; as a powder or granules; as a solution or a suspension inan aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oilemulsion.

In general, the formulations are prepared by uniformly and intimatelyadmixing the active PPAR activator and/or benzoquinone with a liquid orfinely divided solid carrier, or both, and then, if necessary, shapingthe product. For example, a tablet may be prepared by compressing ormoulding a powder or granules of the PPAR activator and/or benzoquinoneoptionally with one or more accessory ingredients. Compressed tabletsmay be prepared by compressing, in a suitable machine, the compound in afree-flowing form, such as a powder or granules optionally mixed with abinder, lubricant, inert diluent and/or surface active/dispersingagent(s). Moulded tablets may be made by moulding, in a suitablemachine, the powdered compound moistened with an inert liquid diluent.

Formulations suitable for buccal (sub-lingual) administration includelozenges comprising a PPAR activator and/or benzoquinone in a flavouredbase, usually sucrose and acacia or tragacanth, and pastilles comprisingthe activator in an inert base such as gelatin and glycerin or sucroseand acacia.

Formulations of the present invention suitable for parenteraladministration conveniently comprise sterile aqueous preparations of aPPAR activator and/or benzoquinone, preferably isotonic with the bloodof the intended recipient. These preparations are preferablyadministered intravenously, although administration may also be effectedby means of subcutaneous, intramuscular, or intradermal injection. Suchpreparations may conveniently be prepared by admixing the activator withwater and rendering the resulting solution sterile and isotonic with theblood. Injectable compositions according to the invention will generallycontain from 0.1 to 5% w/w of the activator and 0.1 to 5% w/w of thebenzoquinone.

Formulations suitable for rectal administration are preferably presentedas unit-dose suppositories. These may be prepared by admixing a PPARactivator and/or benzoquinone with one or more conventional solidcarriers, for example, cocoa butter, and then shaping the resultingmixture.

Formulations suitable for topical application to the skin preferablytake the form of an ointment, cream, lotion, paste, gel, spray, aerosol,or oil. Carriers which may be used include vaseline, lanolin,polyethylene glycols, alcohols, and combinations of two or more thereof.The PPAR activator and/or benzoquinone are generally present at aconcentration of from 0.1 to 15% w/w of the composition, for example,from 0.5 to 2%.

Preferably, the PPAR activator, such as fenofibrate is co-micronisedwith a solid surfactant (for example as described in AU-A-614577). Aparticularly preferred solid surfactant is sodium lauryl sulphate.Typically, the solid surfactant is used in an amount of from 1 to 4%.

The PPAR activator and benzoquinone may be administered separately,sequentially or simultaneously (such as when administered as acomposition comprising both the PPAR activator and a benzoquinone).

In addition, it may also be desirable to administer, in addition to thePPAR activator and/or benzoquinone according to the present invention,further components, such as pharmaceutically active compounds thatimprove vascular condition. As specific examples, it may be desirable toadminister aspirin, an antiotensin converting enzyme inhibitor and/orora calcium channel blocker to the patients before or during treatmentwith the PPAR activator and benzoquinone.

Therapeutic Uses

The mechanism of the improvement of vascular function with thecombination of a PPAR activator such as fenofibrate and a benzoquinonesuch as CoQ₁₀ is likely to be due to a direct effect on the vascularwall, independent to a large extent from the lipid lowering effects ofthe PPAR activator. This synergy could be explained at least in part byeither an interaction with the formation, diffusion or action ofendogenous NO or endothelium-derived hyperpolarizing factor remainspossible. This is supported by the findings with acetylcholine (Ach),sodium nitroprusside (SNP) and the co-infusion ofACh+N^(G)-monomethyl-L-arginine (L-NMMA) in the setting of aspirintherapy. Pretreatment with aspirin also simulates best clinical practiceof preventive medicine, given that aspirin has been shown to diminishcardiovascular events in patients with and without diabetes.

The improvement of endothelial dysfunction provided by the combinationof a PPAR activator such as fenofibrate and a benzoquinone such as CoQ₁₀constitutes a new therapeutic approach which is easy to implement.

Beyond the synergy demonstrated herein with a combination of fenofibrateand coenzyme Q₁₀, similar effects could be obtained with a combinationof other benzoquinone antioxidants (or their precursors) and otherfibrates or PPAR activators which share with fenofibrate an effect onthe expression of multiple genes involved in atherosclerosis, lipidmetabolism and regulation of vascular wall function.

Thus, a combination of a PPAR activator and a benzoquinone may be usedto treat or prevent disorders characterised by endothelial dysfunction,or an increased risk of endothelial dysfunctions. Examples of suchdisorders include cardiovascular events, cardiovascular disease,hypertension, stroke, myocardial infarction, peripheral vasculardisease, angina pectoris, cardiac failure, diastolic and/or systolicventricular dysfunction, macro and microangiopathy in patients withdiabetes, and tissue damage related to ischemia and reperfusion. Inparticular, a combination of a PPAR activator and a benzoquinone may beused to treat patients with type 2 diabetes.

More specifically, the physiological effects associated with theadministration of a combination of a PPAR activator and a benzoquinonemay result in one or more of the following: improved vessel tone,reduced blood clotting, reduced platelet aggregation, reduced bloodpressure and increased blood flow to the heart, reduced smooth musclecell proliferation and inhibition of leucocyte chemotaxis.

Accordingly the present invention also provides a method of improvingvessel tone, reducing blood clotting, reducing platelet aggregation,reducing blood pressure and increasing blood flow to the heart, reducingsmooth muscle cell proliferation, and/or inhibiting leucocyte chemotaxisin a patient which method comprises administering to said patient aneffective amount of a PPAR activator and a benzoquinone.

Vessel tone, platelet aggregation, blood pressure and blood flow, smoothcell proliferation and leucocyte chemotaxis may be measured usingstandard techniques prior to and during treatment to determine whetherthe PPAR activator and a benzoquinone are achieving the desired effect(see for example Furchgott, 1980; Garg, 1989; Radomski, 1987 andMoncada, 1991).

The present invention will now be described further by way of exampleswhich are intended to be illustrative only and non-limiting.

EXAMPLES

Introduction

We have tested a combination of a peroxisome proliferator activatedreceptor (PPAR) activator, namely fenofibrate, and coenzyme Q₁₀ for thetreatment of vascular dysfunction in a randomized clinical trialinvolving patients with type 2 diabetes.

The results obtained demonstrate for the first time a synergism betweena lipid lowering agent (the PPAR activator) and a radical scavenger inreducing vascular dysfunction. This synergistic effect was considered tobe both statistically significant and clinically relevant. Support forthe clinical relevance of these finding is also provided by studiesshowing an association between endothelium dysfunction in peripheralarteries and endothelium dysfunction in the coronary arteries (Anderson,1995; Sax, 1987) as well as longitudinal data showing that endothelialdysfunction predicts future coronary events (Suwaidi, 2000; Schachinger,2000). Compared with treatment with fenofibrate alone, the combinationof fenofibrate with Coenzyme Q improves endothelial dysfunction andpotentially reduces the progression of macro- and microvascular diseasein type 2 diabetes. We would anticipate this to result in improvementswith respect to coronary heart disease, peripheral vascular disease,ischemic stroke, renal disease and retinopathy in diabetes patients andby extension, to subjects with insulin resistance, hypertension andobesity.

Study Design and Methods:

Eighty dyslipidaemic patients with well controlled type 2 diabetes wererandomised double-blind in a 2×2 factorial study to receive fenofibrate(F), CoQ₁₀ (Q), fenofibrate and CoQ₁₀ (FQ), or placebo (P) for 12 weeks.Male or female patients aged less than 70 years and without severeobesity (Body Mass Index below 35 kg/m²) were included after a 6 weekrun-in period if they had Haemoglobin A1c below 9%, total cholesterolbelow 6.5 mmol/l and either triglyceride above 1.8 mmol/l or HDLcholesterol below 1 mmol/l. The two therapeutic agents alone or incombination were given each as 200 mg once daily. Capsules identical inappearance to each agent but containing placebo were given to maintainthe double-blind nature of the study.

Evaluations of vascular function were carried out by bilateral venousocclusion plethysmography at weeks 0 and 12. These consisted of serialmeasurements of forearm blood flow before and after intra-brachialartery infusion of acetylcholine (ACh 7.5, 15 and 30 μg/min), sodiumnitroprusside (SNP 1.5, 3 and 10 μg/min) and N^(G)-monomethyl-L-arginine(L-NMMA 4 μmol/l). These tests were performed after discontinuation ofagents that might have changed vascular function such as ACE inhibitorsand calcium channel blockers. Pretreatment with acetylsalicylic acid(aspirin) (650 mg daily taken orally) was given for one week to blockprostacyclin and thromboxane generation.

Plethysmography studies were performed during a 5 minute infusion ofeach agent, diluted in saline and infused at a rate of 1 mL/min into thebrachial artery of the non-dominant arm via a thin plastic cannula Eachinfusion of vasoactive agents was preceded by a period of salineinfusion. Bilateral forearm blood flow was measured simultaneously at 15second intervals for the final two minutes of each 5 minute infusionperiod employing mercury-in-silastic strain gauges. During measurementshands were excluded from the circulation by inflation of wrist cuffs to200 mmHg and venous occlusion was obtained by cyclical inflation ofupper arm cuffs to 40 mmHg. Results were expressed as the area under thecurve (AUC) of the percent increase in forearm blood flow ratio (infusedarm versus control arm) to account for any systemic effect of thesevasoactive drugs. The AUC provided integration over time of the doseresponse to the agent used. AUC for percent change in blood flow to AChwas a priori described as the primary efficacy criterion in this trial.

Statistical analyses were performed using 2 by 2 analysis of varianceusing SPSS package

Results:

Out of the 80 patients randomised, 77 completed the 12 week treatmentperiod and paired blood flow data were available in 67; Threewithdrawals occurred because of incidental medical conditions and oneallergy to fenofibrate. 10 patients refused a second cannulation orcould not be cannulated satisfactorily. The four groups were wellmatched in terms of baseline characteristics as shown on Table 1. Only 8patients were on oral hypoglycaemic agents, none were on insulin. Theypresented with good diabetic control and with the typicalcharacteristics of diabetic dyslipidemia.

TABLE 1 Patient characteristics: mean or distribution PP PQ PF FQ groupgroup group group Gender (M/F) 14 M/5 F 18 M/2 F 14 M/5 F 14 M/5 F Age(years) 55 53 54 52 BMI (kg/m²) 31.0 29.9 30.0 30.6 BP (mm Hg) 137/78128/76 131/74 132/77 HbA1c (%) 6.3 6.9 7.1 7.5 TC (mmol/l) 5.36 5.295.54 5.25 TG (mmol/l) 2.44 2.19 2.61 2.98 HDL-C (mmol/l) 1.02 0.95 0.950.93 Key: P = Placebo; F = Fenofibrate; Q = CoQ₁₀ BMI = body mass index;HbA1c = glycosylated hemoglobin A1c TC = total cholesterol; TG =Triglyceride; HDL-C = high density lipoprotein cholesterol

Table 2 shows the main study results where changes in treatment arepresented in a 2×2 table consistent with the factorial study design.

TABLE 2 mean changes and their 95% confidence interval on treatment(Week 12-Week 0) in acetylcholine AUC percent increase in forearm bloodflow ratio P Q F− v F+ [95% CI] P −23% [−143% to −7% [−119% −15% [−97%to 66%] 95%] to 105%] F 131% [8% to 253%] 419% [287% to 275% [185% to550%] 365%] Q− v Q+ 53% [−31% to 206% [119% to 138%] 192% ] Key: P =Placebo; F = Fenofibrate; Q = CoQ₁₀ Analysis of variance Interaction p =0.029, F effect p = 0.0001, Q effect p = 0.015

The combined effect of fenofibrate and CoQ₁₀ treatment led to a 419%increase in forearm blood flow ratio, fenofibrate alone to a 131%increase while there was no change with CoQ₁₀ alone or placebo. Thus,there was a clear synergism between fenofibrate and CoQ₁₀ as evidencedby a significant interaction effect (p=0.029). Changes versus baselinewere significant for the fenofibrate alone and the fenofibrate +CoQ₁₀groups but when the 4 groups were compared with each other only thecombination treatment was significantly different from the 3 othergroups (see FIG. 1)

When the vasodilatory response to ACh was reduced by co-infusion ofL-NMMA similar results with a synergism between fenofibrate and coenzymeQ₁₀ were observed (see Table 3).

TABLE 3 mean changes and their 95% confidence interval on treatment(Week 12-Week 0) in acetylcholine percent increase in forearm blood flowratio with co-infusion of L-NMMA P Q F− v F+ [95% CI] P 58%[−2% to 118%]−21% [−76% to 18% [−22% to 59% ] 33%] F 47%[−10% to 47%] 118% [53% to83% [39% to 126%] 183%] Q− v Q+ 53% [11% to 94%] 48% [6% to 90%] Key: P= Placebo; F = Fenofibrate; Q = CoQ₁₀ Analysis of variance Interaction p= 0.015, F effect p = 0.034, Q effect p = 0.884

TABLE 4 Mean changes and their 95% confidence interval on treatment(Week 12-week 0) in sodium nitroprusside AUC percent increase in forearmblood flow ratio (see Figure 2) P Q F− v F+ [95% CI] P −240%[−632% to50% [−641% to −95%[−503% 152%] 740%] to 313%] F −11%[−521% to 1594%[728%to 792% [352% to 500%] 2461%] 1232%] Q− v −126% [−841% to 822% [399% toQ+ 361%] 1245%] Key: P = Placebo; F = Fenofibrate; Q = CoQ₁₀ Analysis ofvariance Interaction p = 0.032, F effect p = 0.004, Q effect p = 0.002

These improvements in vascular endothelium function were not explainedby any interaction between fenofibrate and CoQ₁₀ on the lipid modifyingproperties of fenofibrate; an increase in HDL-cholesterol, a decrease intotal cholesterol, LDL-cholesterol, triglyceride or fibrinogen.Furthermore plasma levels of CoQ₁₀ after treatment did not differbetween the CoQ₁₀ alone and the combination groups. There was no changein diabetes control or blood pressure measurements with the combinationof fenofibrate and CoQ₁₀. CoQ₁₀ alone had no lipid lowering effects.

All publications mentioned in the above specification are hereinincorporated by reference.

Various modifications and variations of the described methods and systemof the invention will be apparent to those skilled in the art withoutdeparting from the scope and spirit of the invention. Although theinvention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are apparent to those skilled in molecular biology orrelated fields are intended to be within the scope of the invention.

REFERENCES

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1. A method for treating a disorder characterized by endothelialdysfunction in a mammal, the method comprising: administering daily tothe mammal a composition comprising: about 200 mg of fenofibrate or apharmaceutically acceptable salt thereof; and about 200 mg of coenzymeQ₁₀ or a pharmaceutically acceptable salt thereof; the disorder beingcardiovascular disease, hypertension, stroke, myocardial infarction,peripheral vascular disease, macro angiopathy in the mammal withdiabetes, or micro angiopathy in the mammal with diabetes.
 2. The methodof claim 1, comprising administering daily to the mammal a compositioncomprising: 200 mg of fenofibrate or a pharmaceutically acceptable saltthereof; and 200 mg of coenzyme Q₁₀ or a pharmaceutically acceptablesalt thereof.